During the summers of 2002 and 2004, nine people contracted tick-borne relapsing fever on Wild Horse Island in southwest Flathead Lake, Lake County, Montana (Schwan et al, 2003, Emerg. Infect. Dis; Schwan et al., 2007; Emerg. Infect. Dis). Investigations by our group associated with these outbreaks resulted in the first isolations of Borrelia hermsii and the first collection of its tick vector, Ornithodoros hermsi, in Montana. With the emergence of human relapsing fever cases in western Montana, we had the opportunity to explore the role of local vertebrate hosts at maintaining the spirochete in nature. By establishing additional isolates of the spirochete, we were also hoping to determine the genetic diversity and host associations at specific locations. For example, during the human outbreaks on Wild Horse Island we found two diverse genomic groups of B. hermsii infecting two individuals that slept in the same bed (Schwan et al., 2007; Emerg. Infect. Dis). Thus, additional work in the field was intended to complement laboratory investigations examining the possible maintenance of diverse genetic types of spirochetes in vertebrates and ticks. During the summer of 2010 we completed our three year study at six field sites including Wild Horse Island, Melita Island, Cedar Island, Yellow Bay where the University of Montana maintains its Flathead Lake Biological Research Station, on the mountain slope east of Flathead Lake, and at the Polson Big Arm area. Our three years of fieldwork resulted in executing 14,336 trap nights and capturing 681 animals. Of these animals, 666 individuals provided blood that was analyzed for current or past infections with Borrelia hermsii. We captured 11 species of small mammals, including pine squirrels (Tamiasciuris hudsonicus), deer mice (Peromyscus maniculatus), 2 species of chipmunks (Tamias ruficaudus and T. amoenus), flying squirrels (Glaucomys sabrinus), and smaller species of mice and shrews. Nineteen pine squirrels had active spirochete infections when captured and we used laboratory mice to amplify the spirochetes in vivo and successfully established 12 isolates for further analysis. The prevalence of B. hermsii antibodies in the pine squirrels was high at 79.5% (128/161) serum samples positive by Western blot analysis. On Wild Horse Island, nearly all (95%; 39/41) of the pine squirrels were seropositive while at Yellow Bay, 68% (50/74) of the squirrels showed previous infections. Only one of the 366 deer mice had an active spirochetemia and a seroprevalence of 5.5% (20/366). Nearly all of the flying squirrels were seropositive (91%; 10/11) and two species of chipmunks were seropositive that demonstrated prior infections with a seroprevalence combined of 47% (23/49). Cumulatively, we also collected 31 ticks, Ornithodoros hermsi, from pine squirrel nests. These ticks were fed individually on mice in the laboratory and 13 of them transmitted spirochetes. From these infected mice, we established nine new isolates of B. hermsii. By the end of our 2010 field season, we established 21 isolates of B. hermsii from squirrels and ticks. With our 6 human isolates from patients infected on Wild Horse Island, these 27 isolates provide the largest number of B. hermsii isolates from any endemic focus of tick-borne relapsing fever. We determined the DNA sequences for the glpQ, flaB, gyrB and 16S rRNA genes, which identified the two genomic groups of B. hermsii we have found elsewhere in western North America. Most of the genetic diversity observed in these spirochetes from throughout North America was observed among the spirochetes isolated at Flathead Lake. And a third genomic group emerged from our work not defined previously. Our efforts demonstrated that on Wild Horse Island, pine squirrels were the premier vertebrate host from maintaining the tick and spirochete populations. Our efforts also demonstrated for the first time anywhere that pine squirrels and deer mice are hosts for B. hermsii in nature.